WO2003104804A1

WO2003104804A1 - Methods of searching for substance having antidiabetic effect

Info

Publication number: WO2003104804A1
Application number: PCT/JP2003/007351
Authority: WO
Inventors: 中西　聡; 矢野　浩史; 聖寿森; 譲松田
Original assignee: 協和醱酵工業株式会社
Priority date: 2002-06-10
Filing date: 2003-06-10
Publication date: 2003-12-18
Also published as: AU2003244107A1; JPWO2003104804A1

Abstract

It is intended to provide a method of searching for a substance having an antidiabetic effect which comprises [1] the step of bringing a substance inhibiting the activity of the potassium-ATP channel of pancreatic β cells into contact with pancreatic β cells in the presence of a test substance and measuring the cell response of the cells upon the contact, [2] the step of bringing the above substance inhibiting the activity of the potassium-ATP channel of pancreatic β cells into contact with the above cells in the absence of a test substance and measuring the cell response of the cells upon the contact, and [3] the step of comparing the obtained data and thus selecting a test substance altering the cell response of the above cells; and a method of searching for a substance having an antidiabetic effect which comprises [1] the step of bringing a test substance into contact with pancreatic β cells having been desensitized to a compound having a sulfonylurea structure at a high glucose concentration and measuring the cell response of the cells upon the contact, [2] the step of measuring the cell response of the above cells at a high glucose concentration, and [3] the step of comparing the obtained data and thus selecting a substance altering the cell response of the above cells from among the test substances.

Description

Specification

Method for searching for substance having anti-diabetic action

Technical field

The present invention relates to a method for searching for a substance having an antidiabetic action.

Background art

Compounds having a sulfonyl-rea structure (hereinafter abbreviated as sulfonyl-rea compounds)

) Is used as a therapeutic agent for type 2 diabetes, which promotes insulin secretion from knee / cells and lowers blood sugar levels.There are many types of sulfonyls with different levels of hypoglycemic action and duration of action.ゥ Rare compounds are known [Joslin's Diabetes Mellitus, Lea and Febiger Ltd. (1994)].

Nateglinide is a compound that does not have a sulfonylurea structure, but like sulfonylurea compounds, is known as a compound that inhibits the activity of calixuma ATP channels and promotes secretin [Journal of Pharmacology & Experimental Therapeutics] , 293, 444 (2000)].

Sulfonylurea compounds have been shown to have common side effects and complications, as well as ineffectiveness against sulfonylurea compounds [Joslin's Diabetes Mellitus, Lea and Febiger Ltd. (1994)]. A major complication with the use of sulfonylprea compounds is hypoglycemia [Br. Med. J. Clin. Res. Ed., 296, 949 (1988) Joslin's Diabetes Mellitus, Lea and Febiger Ltd. (1994), Acta. Med. Scand. Suppl., 605, 7 (1977)].

It is also known that sulfonylurea compounds are ineffective (primary ineffective) in about 20% of patients diagnosed with type 2 diabetes [Multicenter study,, 404 (1983), Berhhard, H Diabetes, 14, 59 (1965), Joslin's Diabetes Mellitus, Lea and Febiger Ltd. (1994)].

Furthermore, even in patients who had good control of blood glucose with the sulfonylurea compound at the beginning of treatment, the dose of the sulfonylurea compound needed to control blood glucose increased with continued treatment [Joslin ³ s Diabetes Mellitus, Lea and Febiger Ltd. (1994)], and sulfonylurea compounds are known to be ineffective (secondary ineffectiveness) in 3-5% of the patients [Diabetes Care, 9, 129 (1986 ), Metabolism, 15, 957 (1966), Non-insulin-dependent diabetes mellitus, 52 (1989)].

In some cases, continued glycemic control with sulfonylprea compounds leads to remissions in which near-normal glycemic control can be achieved even if treatment is interrupted, but the number is very small (Eur. J. Clin. Pharmacol., 22, 459 (1982), Medicine, 69 3 176 (1990)] since, type 2 diabetes most, throughout life from being diagnosed with diabetes, must undergo some treatment is there.

Therefore, there is a demand for the development of a therapeutic agent for diabetes which is effective even for patients who have become secondarily ineffective against the sulfonyliderea compound. -It is known that in patients who have failed secondary to sulfonylurea compounds, not only administration of sulfonylurea compounds but also promotion of insulin secretion against glucose tolerance [Diabetes, 35] , 1314 (1986)].

—Although it is known that insulin secretion is promoted in response to glucagon even in patients with such secondary ineffectiveness [Diabetes,, 1314 (1986)] Even in the secondarily disabled kidneys, there are insulins in cells that secrete insulin in response to a compound that promotes insulin secretion by a mechanism different from sulfonylurea compounds. Have been.

Similar phenomena can be observed by ex vivo or in vitro experiments. That is, the high-concentration Langerhans islet, which was prepared from the rat to which the sulfonylurea compound was administered for a long time, or the long-ranged Langerhans islet, which was pre-cultured for a long time in the medium containing the sulfonylurea compound. Insulin secretion ability for glucose or sulfonyl-rea compound decreases, but insulin reacts with a compound that promotes insulin secretion by a mechanism different from sulfonyl-rea compound such as glucagon-theophylline. It is known to be secreted [Diabetes, 19, 603 (1970), Endocrinology, 113, 1972 (1983), Diabetologia, 10, 27 (1974), Metabolism, 26, 9 (1977), Endocrinology, 131, 1815 (1992), J. Clin. Endocrinol. Metabol., 74, 790 (1992), Endocrinol. Invest., 14, 287 (1991), Endocrinology, 125, 281 (1989)].

This phenomenon is known as desensitization of the kidney / cells to the sulfonylurea compound by long-term treatment with the sulfonylurea compound. It is considered to be at least one of the causes of secondary ineffectiveness for sulfonylprea compounds [Joslin ⁵ s Diabetes Mellitus, Lea and Febiger Ltd. (1994)] ₀ There is a need for a method for efficiently searching for a compound that acts on such desensitized lignin / cells and has the effect of promoting insulin secretion.

A substance having a function of promoting insulin secretion, comprising selecting a substance having a function of promoting insulin secretion of a cell under a low glucose concentration by using Teng 5 cells desensitized to a sulfonylprea compound. Search methods are known

[Biochemical Pharmacology, 61, 527 (2001)]. However, since the substance obtained by the search method promotes insulin secretion even in the presence of low concentration of glucose, it is possible that hypoglycemia may be caused.

There is no known method to efficiently search for a substance that is effective for patients who are ineffective against sulfonylurea compounds and that has an antidiabetic effect that does not cause hypoglycemia, which is one of the side effects of sulfonylurea compounds. Not been.

Disclosure of the invention

An object of the present invention is to provide a method for searching for a substance having an antidiabetic effect that is effective even for patients who have become secondarily ineffective against sulfonylurea compounds.

The present invention relates to the following (1) to (14).

(1) [1] In the presence of a test substance, measure the cellular response of the knee / cell when contacting a substance that inhibits the activity of the potassium-ATP channel with the kidney / cell. [2] a step of measuring the cellular response of the cell when the cell is brought into contact with a substance that inhibits the potassium-ATP channel activity of the cell in the absence of a test substance; Comparing the measured values and selecting a test substance that changes the cell response of the cell, a method for searching for a substance having an antidiabetic action.

(2) [1] a step of measuring the cellular response of a Tengen /? Cell desensitized to a compound having a sulfonylprea structure under a high glucose concentration in the presence of a test substance; In the presence of the cell, measuring the cell response under the high glucose concentration; [3] comparing the measured values and selecting a test substance that changes the cell response of the cell. A method for searching for a substance having a diabetic action. (3) The method according to (1) above, wherein the substance inhibiting the potassium-ATP channel activity of the spleen cells is a compound having a sulfonylprea structure or nateglinide.

(4) The method according to any one of (1) to (3) above, wherein the substance having an antidiabetic action is a substance having an insulin secretion promoting activity.

(5) The cell response according to any of (1) to (4) above, wherein the cellular response is insulin secretion from S-extracted // cells, and the change in cellular response is promotion of insulin secretion from knee // cells. One search method.

(6) The cell response is a cell response related to the promotion of insulin secretion of Teng // cells, and the change of the cell response is an increase in the cell response related to promotion of insulin secretion of Teng /? Cells. One of the search methods (1) to (4).

(7) The cell response is a cell response related to the suppression of insulin secretion of the hepatic cells, and the change in the cell response is a decrease in the cell response related to the suppression of insulin secretion of the five viable cells. ) To (4).

(8) The method according to (6) above, wherein the cellular response related to the promotion of insulin secretion is cell membrane capacity, intracellular calcium ion concentration, cell membrane potential, intracellular cyclic AMP concentration, or calcium ion channel activity.

(9) The method according to the above (7), wherein the cellular response related to the suppression of insulin secretion is intracellular rheumatoid ion concentration or potassium ion channel activity.

(10) The search method according to any one of the above (2) to (9), wherein the compound having a sulfonylprea structure is glibenclamide or tolbutamide.

(11) Teng cells that have been desensitized to a compound having a sulfonylprea structure are desensitized to the parent compound by their ability to secrete insulin in the presence of a high concentration of glucose, or by a cellular response involved in promoting insulin secretion. The search method according to any one of the above (2) and (4) to (9), wherein the number of cells is smaller than that of non-produced knee cells.

(12) Neural 5 cells desensitized to a compound having a sulfonylprea structure did not desensitize the parental compound to a cell response related to suppression of insulin secretion in the presence of high concentration of glucose. The cells that are increasing compared to the kidney /? Cells The search method according to any one of 2) and (4) to (9).

(13) The method for searching according to any one of (1) to (9) above, wherein the tongue /? Cells are MIN6 cells.

(14) The search method according to any one of (3) to (9) above, wherein the germ cells are MIN6 cells and the compound having a sulfonylprea structure is glibenclamide or tolptamide.

The brain cells used in the invention according to the above (1) may be any compound having a sulfonyl-peryl structure (hereinafter, referred to as a sulfonyl-peryl compound) or any cell that responds to nateglinide. Can also be used, for example, a ^^^ cell prepared from a vicinal cell, or a 臓 // cell line derived from a vicinal cell. As a cell line, RINm5F cells [Diabetes, 31, 521 (1982)], BRIN-BD11 cells [Diabetes, 45, 1132 (1996)], INS-1 cells [Endocrinology, Ya, 167 (1992)] Natl. Acad. Sci. USA., 85, 9037 (1988)] and MIN6 cells [Endocrinology, 127, 126 (1990)].

The thyroid cells desensitized to the sulfonylurea compound used in the invention according to the above (2) include, when brought into contact with the sulfonylurea compound, secretion of diinsulin induced by the same concentration of the compound. A cell whose activity is lower than that of a knee ^ cell which has not been desensitized to the parent compound, a cell response relating to promotion of dinsulin secretion, has not been desensitized to the parent compound, Cells that are less than the cells of the brain, or cells whose cell response related to the suppression of insulin secretion is greater than those of the kidney cells that have not been desensitized to the parent compound, etc. Preferably, there can be mentioned cells in which the trait in the desensitized state is equivalent to that of the mouse β cells desensitized to the sulfonylurea compound prepared from the viscera.

Examples of the characteristics of T-cell β cells desensitized to a sulfonylurea compound prepared from the kidney include, for example, insulin secretion activity desensitized in the presence of both low and high concentrations of glucose. Characters that are lower than before cropping can be mentioned. As the concentration of the above-mentioned high concentration of glucose, for example, 5-20 ol / L, preferably 8-18 ol / L, more preferably 9-16 ol / L, and still more preferably 12- ol / L, particularly preferably 14.5 t ol / L. The concentration may be less than 5 mol / L, preferably 1 mol / L or more to less than 5 mol / L, more preferably 2 mol / L or more to less than 5 mmol / L.

The above-mentioned parent strain refers to the original Teng / T cells subjected to the production of Teng / T cells desensitized to the sulfonylurea compound described below. Spleen cells prepared from the spleen, and TIN /? Cell-derived virulent /? Cell lines such as RINm5F cells, BMN-BD cells, INS-1 cells,? TC cells, and MIN6 cells And preferably MIN6 cells.

Spleen β cells desensitized to the sulfonylurea compound can be obtained by culturing the kidney cells in the presence of the sulfonylurea compound.

As the knee / cells used for the culture, spleen / 5 cells prepared from the above-described brain, MNm5F cells, BMN-BD11 cells, INS-1 cells, and TCTC cells which are five cell lines of the spleen And MIN6 cells, and preferably MIN6 cells.

The fact that the cells obtained by culturing the knee cells in the presence of the sulfonylurea compound desensitized the ligated compound means that the cultured cells of the kidney / cells cultured in the presence of the compound were desensitized. When stimulated with a compound, cells that secrete a small amount of diinsulin and are involved in the promotion of diinsulin secretion are less than those obtained by stimulating the cultured cells of the parent strain, Tengella ^ cells, cultured in the absence of the compound, with the compound. It can be confirmed that the response is small, or that ③ the cell response to suppression of insulin secretion and the response is large.

The sulfonylurea compound used in the present invention may be any compound as long as it is a sulfonylurea compound having an antidiabetic action, and examples thereof include glibenclamide and tolptamide.

Nateglinide used in the present invention also includes a derivative of nateglinide and a compound having an activity to promote the secretion of insulin by knee cells.

The concentration of the sulfonylurea compound and nateglinide used in the method of the present invention according to the above (1) is such that the sulfonylurea compound or nateglinide used is the same as the concentration of the sulfonylurea compound or nateglinide used in the method of the present invention. ^ Any concentration may be used as long as it produces a cell response in the cells and does not show toxicity to the cells.

As the concentration, for example, glibenclamide is used as a sulfonylprea compound. O, the concentration can be increased from 0.1 to 1000 nmol / L, preferably from 10 to 500 dishes ol / L.o In addition, when tolbulamide is used as the sulfonyl perrea compound, the concentration is 50 to 500 mol / L, Preferably, the concentration can be 100 to 200 mol / L. When nateglinide is used, the concentration may be 1 to 100 mol / L, preferably 5 to 20 mol / L.

The concentration of the sulfonylurea compound when desensitizing the kidney cells using the sulfonylurea compound depends on the concentration of the sulfonylurea compound used and the desensitization of the knee / cell used. Any concentration may be used as long as it does not exhibit toxicity to the cells.

As the concentration, for example, when glibenclamide is used as the sulfonylprea compound, a concentration of 0.1 to 1000 nmol / L, preferably 10 to 500 ol / L can be mentioned. When tolbulamide is used as the sulfonylurea compound, the concentration can be increased from 50 to 500 mol / L, preferably from 100 to 20θΛηο1Α.

Spleen cells used in the search method of the present invention according to the above (1) can be prepared by culturing the cells in a medium suitable for the growth of the cells.

The culture medium and culture conditions for culturing the knee / cell used in the search method of the present invention according to the above (2) are a medium suitable for the growth of the cells, and a sulfonylprea compound is added to the medium. Any culture medium or culture condition may be used as long as the culture conditions are such that the cells are desensitized to the compound when added and cultured.

Examples of the medium include a DMEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) to which serum components such as fetal bovine serum are added. The culturing conditions include the conditions of culturing in the presence of 4 to 6%, preferably 5% carbon dioxide at 36 to 38 ° C, preferably 37 ° C for 6 to 72 hours, preferably 18 hours. be able to.

When the substance that inhibits the activity of the ATP channel in the appropriate medium or buffer in the presence of the test substance in the presence of the test substance, Cells of the cells when the cells are contacted with the substance that inhibits the activity of the ATP channel in the medium or the buffer in the absence of the test substance. Obtaining a substance having an anti-diabetic effect by measuring and comparing responses and selecting a test substance that changes the cell response of the cell from the test substance. 351 can be done.

The medium or buffer used for measuring the above-described cell response may be a medium or buffer of any composition as long as the medium or buffer can measure the cell response.

Examples of the medium include a DMEM medium, and examples of the buffer include a HEPES buffer containing a physiological salt.

The medium or buffer used to measure the cellular response may contain glucose and / or glucosyl as long as the concentration of the cellular response can be measured depending on the presence or absence of the test substance. In this case, the preferred concentration is 1 to 20 mol / L, more preferably 1 to 5 IMO1 / L, and still more preferably 2 to 4 mmol / L.

In addition, in the presence of the test substance, the cell response of the mouse β cells desensitized to the sulfonylurea compound in a suitable medium or buffer containing a high concentration of glucose, and in the absence of the test substance, By measuring and comparing the cell response of the cells in the medium or the buffer and selecting a test substance that changes the cell response of the cells, a substance having an antidiabetic effect can be obtained.

The above cell response can be measured by any method capable of measuring the cell response of knee / cells desensitized to a sulfonylurea compound in the presence of high concentration of glucose. For example, for example, after culturing the cells in a medium containing a high concentration of glucose to which a test substance has been added for a certain period of time, or in a buffer containing a high concentration of glucose, the cultured cells and the test substance are mixed. After contacting the cells for a certain period of time, the cell response can be measured.

The medium or buffer for measuring the cell response may be a medium or a buffer of any composition as long as the medium or the buffer can be measured. Examples thereof include a DMEM medium, and a buffer such as a HEPES buffer containing a physiological salt.

A high concentration of glucose in a medium or a buffer solution for measuring cell response refers to the presence of that concentration of glucose in the presence of the test substance and a sulfonylurea compound in the desensitized arm /? Cells. A glucose concentration that is high enough to detect the insulin secretion or cell response of the cells caused by the contact, and the glucose concentration is, for example, 5 to 20 employment ol / L, Preferably 8-18 ol / L, more preferably 9- 16 ol / L ヽ More preferably 12 ~: L5 mmol / L, particularly preferably 14.5 ol / L.

The cell response to be measured in the search method of the present invention may be any cell response as long as it is a cell response related to the suppression of the disease state of diabetes. Examples of the cell response related to the suppression of the disease state of diabetes include: And secretory insulin, and cellular responses involved in insulin secretion.

Examples of the cellular response related to insulin secretion include an increase in a cellular response related to promotion of insulin secretion and a decrease in a cellular response related to suppression of insulin secretion.

Increases in cellular responses related to the promotion of insulin secretion include, for example, an increase in cell monthly capacity [J. Physiol., 472, 665 (1993)], an increase in intracellular calcium ion concentration, an increase in cell membrane potential, Increase in intracellular cyclic AMP concentration, activation of calcium ion channel activity, increase in intracellular potassium ion concentration [all described in Joslin's Diabetes Mellitus, Lea and Febiger Ltd. (1994)].

Examples of the decrease in the cellular response related to suppression of insulin secretion include a decrease in potassium ion concentration and a decrease in potassium ion channel activity [both described in Joslin's Diabetes Mellitus, Lea and Febiger Ltd. (1994)]. Insulin secretion that can be mentioned can be confirmed by detecting and quantifying insulin secreted into a culture solution or a buffer solution. As a method for detecting and quantifying insulin, any known method can be used, and for example, a radioimmunoassay method using a radiolabeled anti-insulin antibody can be used.

The fact that the substance selected by the method of the present invention does not promote the secretion of insulin by visceral cells and knee / cells desensitized to a sulfonylurea compound in the presence of low concentration of glucose, To promote insulin secretion and insulin secretion when contacting the dermis with cells in the presence of low-concentration glucose in a suitable culture medium or buffer, when the dermis / cells or sulphonylurea compounds are desensitized to the appropriate amount of medium. It can be confirmed by measuring the cellular response involved or the inhibition of insulin secretion. The low-concentration glucose may include a concentration of less than 5 mol / L, preferably 1 ol / L or more to less than 5 Fiber ol / L, and more preferably 2 ol / L or more to less than 5 ol / L. . The kidney / cells, culture medium, buffer and the like used in the above-mentioned measurement may be any as long as they are used in the above-described search method of the present invention. Insulin secretion, cell response related to promotion of insulin secretion, or cell response related to suppression of insulin secretion can be measured by the methods described above.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the effect of compound 1 on promoting insulin secretion from MIN6 cells in the presence of glibenclamide. The open column only adds 100 nmol / L glibenclamide, the black column adds 100 nmol / L glibenclamide and l〃mol / L compound 1, and the hatched lamb shows 100 nmol / L glibenclamide plus 10 zmol / L. The plot shows the test plot to which glibenclamide was added, and the vertical axis shows the insulin concentration in the supernatant. Error bars indicate the standard error in experiments performed in triplicate.

FIG. 2 is a graph showing the insulin secretion promoting effect of Compound 1 on cells desensitized to glibenclamide. A shows the measurement of insulin secretion in the presence of 2 ol / L of glucose, and B shows the measurement of insulin secretion in the presence of 14.5 liter / L of glucose. The error bars indicate the standard errors of the experiments performed in triplicate. In the column, the white column indicates the group without addition of the test substance, the scattered point indicates the group with added compound 1, the shaded line indicates the group with glibenclamide added, and the vertical axis indicates the amount of insulin that was secreted and remained in the cells after secretion. The ratio of the amount of insulin secreted to the amount of insulin contained in the cells before the secretion test, calculated by adding the amount, is expressed as a percentage (% insulin secretion).

The lines connecting the columns in the figure indicate significant differences between the columns, NS indicates no significant difference, ★ indicates p <0.05, ★★ indicates p <0.01, and ★★★ indicates p <0.001 .

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto.

Example 1 Method for searching for a substance having an insulin secretion promoting action using MIN6 cells (1)

MIN6 cells were cultured in a multi-well plate for cell culture (24-well plate, manufactured by KONING) at 37 ° C, in the presence of 5% carbon dioxide, with 15% fetal bovine serum and 5 g / g L glucose After culturing in a DMEM medium (Dulbecco's Modified Eagle Medium, manufactured by Nissui Pharmaceutical Co., Ltd.) until confluency, HBKRPB buffer containing 2 ol / L glucose (119 ol / L) was used. Shiojiri sodium, 4.74 thigh 01 Shiridani potassium, 2.54 hi ol / L calcium chloride, 1.19 mmol / L magnesium sulfate, 1.19 thigh ol / L potassium dihydrogen phosphate, 10 ol / L 2-[4- ( 2-Hydroxyethyl) -1-piperazinyl] ethanesulfonic acid, 0.1% serum albumin, pH 7.3), and then washed with HBKRPB buffer containing 2 ol / L glucose. Incubated at 45 ° C for 45 minutes. Thereafter, the buffer was added to 1 mL / 100 nmol / L of glibenclamide and 2 ol / L of HBKRPB buffer containing glucose, and 1 / mol / L of the following formula (I) was added to the buffer.

And a solution in which 10 mol / L glibenclamide was added to the buffer solution, and the cells were incubated at 37 ° C for 45 minutes. After sampling, sampling was performed. The supernatant was appropriately diluted with a phosphate buffer containing 0.1% of serum albumin and 0.01% of sodium azide, and used for measurement of insulin concentration.

The insulin concentration of the diluted supernatant was determined based on the radioimmunoassay method using 3- [ ¹²⁵ I] iodotyrosyl ^A14 insulin (Amersham Pharmacia Biotech) and anti-ratinsulin antibody (Cosmo Bio). Rat insulin (Cosmo Bio Inc.) was measured as a standard. Fig. 1 shows the measurement results.

From FIG. 1, it was confirmed that Compound 1 has insulin secretion promoting activity even in the presence of glypenclamide which is a sulfonylurea compound.

Example 2 Method for searching for substance having insulin secretion promoting action using MIN6 cells (2)

Transfer MIN6 cells to a multiwell plate for cell culture (24-well plate, Medium) at 37 ° (Dulbecco's Modified Eagle Medium, containing 15% fetal calf serum and 5 g / L glucose in the presence of 5% CO 2) , And then the medium was replaced with a medium containing 10 nmol / L glibenclamide (manufactured by Sigma), followed by further culturing for 18 hours.

The MIN6 cells pre-cultured in the medium containing glibenclamide in this manner were washed with HBKRPB buffer containing 2 ol / L glucose, and then washed in HBKRPB buffer containing 2 ol / L glucose. And incubated at 37 ° C for 45 minutes.

Thereafter, an insulin secretion test was performed by the following method.

First, add HBKRPB buffer containing 2 ol / L of glucose to 10 mol / L of the following formula (I)

Was replaced with HBKRPB buffer containing compound 1 (WO00 / 01388) and glucose at 2 mol / L, and the cells were treated at 37 ° C. for 15 minutes. The final concentration of glucose was adjusted to 14.5 fol / L by adding 1/9 volume of the above-mentioned exchange solution to HBKRPB buffer containing 127 l / l glucose. Thereafter, the cells were incubated at 37 ° C. for 45 minutes, and the supernatant containing the secreted insulin was sampled. After the insulin secretion test was completed, insulin remaining in the cells was extracted by adding 3 mol / L acetic acid to the cells, and the supernatant was sampled. The supernatant was appropriately diluted with a phosphate buffer containing 0.1% of serum albumin and 0.01% of sodium azide, respectively, and used for measurement of the amount of insulin. The amount of insulin was determined based on the radioimmunoassay method using 3- [ ¹²⁵ I] iodotyrosyl ^A14 insulin (Amersham Pharmacia Biotech) and anti-ratinsulin antibody (Cosmo Bio) and rat insulin (Cosmo Bio). Was measured as a standard. By adding the amount of insulin that remained in the cells after secretion to the amount of insulin that was secreted, the amount of insulin that was contained in the cells before the secretion test was determined. Was.

Insulin secretion% was defined as the percentage of the amount of insulin secreted to the amount of insulin contained in the cells before the secretion test.

FIG. 2 shows that a substance that promotes the secretion of insulin from the hepatic cells desensitized to the sulfonylrea compound by the search method of the present invention can be selected (right of FIG. 2B). Columns). In addition, Compound 1 selected by the method of the present invention is not desensitized to a sulfonylurea compound and has the same insulin secretion-promoting action on knee cells as a sulfonylurea compound. It was confirmed (left-hand ram group in Fig. 2B).

In addition, it was confirmed that Compound 1 did not promote insulin secretion of ligament cells in the presence of low concentration of glucose.

Industrial applicability

According to the present invention, changes in the cellular response of england β cells in the presence of a substance that inhibits the activity of the potassium-ATP channel of england / 勝 cells, and the effects of renocytes /? Cells desensitized to sulfonylurea compounds, Using the change in cell response as an index, it is possible to efficiently search for a substance having an antidiabetic effect that is effective even for patients who are ineffective for sulfonylurea compounds

Claims

The scope of the claims

1. [1] Measuring the cellular response of the kidney cells when a substance that inhibits the activity of potassium-ATP channel in 5 cells of the spleen is brought into contact with Teng cells in the presence of the test substance, [2] Measuring the cell response of the cell when the cell is brought into contact with a substance that inhibits the potassium-ATP channel activity of the cell in the absence of the test substance; [3. Comparing and selecting a test substance that changes the cell response of the cell, the method of searching for a substance having an anti-diabetic action.

2. [1] The step of measuring the cell response of high-concentration glucose cells desensitized to a compound having a sulfonylprea structure in the presence of the test substance under high glucose concentration. [2] In the absence of the test substance, Measuring the cellular response of the cell under the high glucose concentration

[3] A method for searching for a substance having an anti-diabetic effect, comprising: comparing the measured values and selecting a test substance that changes the cellular response of the cell.

3. The search method according to claim 1, wherein the substance that inhibits the activity of the potassium-ATP channel of the five cells of the brain is a compound having a sulfonylurea structure or nateglinide.

4. The search method according to any one of claims 1 to 3, wherein the substance having an antidiabetic action is a substance having an insulin secretion promoting activity.

5. The search according to any one of claims 1 to 4, wherein the cell response is insulin secretion from kidney / cells, and the change in cell response is promotion of insulin secretion from kidney β cells. Method.

6. The cellular response is a cellular response related to the promotion of insulin secretion of the kidney / cell, and the change in the cell response is an increase in the cellular response related to the promotion of insulin secretion of the kidney / cell. Item 5. The search method according to any one of Items 1 to 4.

7. The cell response according to claim 1, wherein the cell response is related to the suppression of insulin secretion of the viable cells, and the change in the cell response is a decrease in the cell response related to suppression of the insulin secretion of the brain cells. 5. The search method according to any one of items 4 to 4.

8. The search method according to claim 6, wherein the cellular response related to promotion of insulin secretion is cell membrane capacity, intracellular calcium ion concentration, cell membrane potential, intracellular cyclic AMP concentration, or calcium ion channel activity.

9. The search method according to claim 7, wherein the cellular response related to the suppression of insulin secretion is intracellular potassium ion concentration or potassium ion channel activity.

10. The search method according to any one of claims 2 to 9, wherein the compound having a sulfonylprea structure is glipenclamide or tolpumidamide.

11. Swelling cells desensitized to a compound having a sulfonylprea structure have an ability to secrete insulin in the presence of a high concentration of glucose, or a cellular response related to promotion of insulin secretion, to the parent compound. 10. The search method according to any one of claims 2 and 4 to 9, wherein the number of cells is decreased as compared with 5 cells of the unsensitized Tengen.

12. Nervous cells that have been desensitized to a compound having a sulfonylprea structure do not desensitize to the parent compound, a cell response related to suppression of insulin secretion in the presence of high concentration of glucose. 10. The search method according to any one of claims 2 and 4 to 9, wherein the number of cells is larger than that of kidney / cells.

13. The search method according to any one of claims 1 to 9, wherein the Tengen /? Cells are MIN6 cells.

14. The search method according to any one of claims 1 to 9, wherein the tongue / cells are MIN6 cells, and the compound having a sulfonylprea structure is glibenclamide or tolptamide.